The adoptive transfer of lymphocytes engineered to recognize tumor cells has shown tremendous promise in patients with relapsed or refractory B cell malignancies. In this approach, patient derived T cells are programmed in vitro with engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs) that have affinity for cancer- or lineage-specific antigens. Subsequent autologous reinfusion of the engineered T cells enables tumor targeting and eradication. More recently, genome editing approaches have been proposed to improve engineered T cell performance by knocking out genes that mediate graft-versus-host disease (for allogeneic transfer), that are involved in tumor-mediated suppression of T cell efficacy, or that are recognized by potent clinically relevant monoclonal antibodies. However, to this point no robust characterization of genome editing efficiency or specificity has been performed in T cells. Much of my postdoctoral work has focused on developing methods to characterize and improve the utility and genome-wide specificity of CRISPR-Cas nucleases, making me uniquely suited to address this largely outstanding question. The primary aims of this proposal are therefore: 1) to characterize and optimize the efficiency and specificity of genome editing in T cells, 2) utilize genome editing to improve processes involved in T cell engineering, and 3) leverage CRISPR-Cas screens to enhance the overall efficacy of engineered T cells. The proposed research will provide considerable insight into the feasibility of implementing genome editing strategies in T cells as a means to improve tumor killing efficacy, persistence, or manufacturing. Significant findings relevant to the fields of cancer immunotherapy, cancer biology, genome editing, and gene therapy are expected. Areas of additional scientific training that will enable successful completion of this proposal are knowledge of T cell biology and immunology, mentorship on in vivo cancer modeling in mice, and experience implementing genome-wide CRISPR-Cas screens. The mentored phase of the award will be supported by Dr. Keith Joung, a world-leader in genome editing technology development, and by Dr. Marcela Maus, an expert in cancer immunotherapy. Dr. Joung, Dr. Maus, and nearly all other senior/key personnel of this project are located at the Massachusetts General Hospital or in the greater Boston scientific community. Professional and career development activities will include training in mentorship, responsible conduct of research, grantsmanship, finance, and conflict of interest among other topics. National and international meetings will continue to be attended to disseminate findings from the proposed research. The academic and professional development environment at the Massachusetts General Hospital and Harvard Medical School, combined with a top level mentorship team and scientific advisory committee, will offer the best opportunity for further training as I transition to independence.